Group B Streptococci have, in recent years, become known as a significant cause of serious neonatal infections. Clinically, two distinct syndromes have been recognized, i.e., "early onset" disease, beginning up to about five days after birth; and "late onset" disease, beginning about ten days to three months after birth.
Early onset disease is characterized by a high incidence of maternal complications. Pneumonia, respiratory distress syndrome, and bacteremia are common; whereas meningitis occurs in slightly less than one-third of the cases. Overall mortality is about 55%.
Late onset disease usually is not associated with maternal complications. About 75% of the late onset disease is meningitis. The infections of late onset disease are generally less severe than those of early onset disease. Overall mortality is about 25%.
Present data indicate that early onset disease occurs in about two per one thousand live births and late onset disease occurs in one per one thousand live births.
Group B Streptococci are classified into five serotypes on the basis of antigenic material extracted from the cell wall of the bacteria by hydrochloric acid. The serotypes are denoted Types Ia, Ib, Ic, II and III. Epidemiological studies to determine the incidence of maternal colonization suggest that as many as 25% of pregnant women harbor Group B Streptococcus as part of their normal vaginal flora. This flora consists of about 18-28% Types I, 34-44% Type II, and 33-37% Type III. Most of the early onset disease is caused by the same serotypes harbored by the mother, implying maternal-fetal transmission.
Group B, Type III Streptococcus strains cause about 80% of early onset meningitis and about 93% of late onset meningitis. Type III also causes about 90% of all late onset Group B Streptococcal infections.
Group B Streptococci cause the formation of antibodies in some women who are carriers of Group B Streptococci or who have had Group B Streptococcal infections. These antibodies are of several immunoglobulin types including the immunoglobulin G (IgG) type. This is manifested by the observation that uninfected neonates born of mothers with Group B Streptococcus antibodies have the same antibodies in their blood. This transplacental transfer of antibodies is a characteristic of IgG antibodies.
For many years a means of preventing Group B Streptococcal infections in pregnant women and neonates have been sought. A vaccine suitable for the purpose of immunizing women of childbearing age, particularly pregnant women, was sought, but until this invention was made, no one has been successful in producing a suitable vaccine, since no one has been able to prepare pure Group B or Type III antigens having the characteristics required for a successful vaccine.
The characteristics of a desirable vaccine against a bacterial disease are as follows:
(a) Immunize selectively against the bacterial antigen to prevent the disease for extended periods of time; PA1 (b) Cause no undesirable reactions; PA1 (c) Be monovalent; and PA1 (d) Require low doses and one or two administrations. PA1 acid casein hydrolyzate adjusted to pH 7: 100 grams PA1 1-glutamine: 6 grams PA1 1-asparagine: 6 grams PA1 monopotassium phosphate: 8.84 grams PA1 dipotassium phosphate: 6.10 grams PA1 ammonium sulfate: 12 grams PA1 1-tryptophan: 2 grams PA1 riboflavin: 16 mg PA1 pantothenic acid: 34.4 mg PA1 thiamine hydrochloride: 16 mg PA1 PABA: 3.2 mg PA1 nicotinamide: 80 mg PA1 biotin: 0.4 mg PA1 folic acid: 4 mg PA1 pyridoxamine: 46 mg PA1 glucose (5% aqueous sol.): 1,000 gm PA1 Na.sub.2 HPO.sub.4 : 252 gm PA1 NaH.sub.2 PO.sub.4 : 164 gm PA1 sodium acetate: 120 gm PA1 cysteine: 13 gm PA1 FeSO.sub.4.7H.sub.2 O: 400 mg PA1 1-cystine: 4 gm PA1 adenine sulfate: 435 mg PA1 guanine hydrochloride: 310 mg PA1 uracil: 250 mg PA1 MgSo.sub.4.7H.sub.2 O: 8,000 mg PA1 NaCl: 400 mg PA1 MnSO.sub.4.H.sub.2 O: 302 mg PA1 sodium citrate: 4.5 gm
In order to accomplish the preceding, it is necessary to obtain pure antigens which are specific to their antibodies and which can be obtained in a form sufficiently pure to cause no side reactions in the patient and of sufficient molecular size to elicit specific antibody production in humans. The antigens as well as the vaccine must be of sufficient purity so that no cross-reactions with human tissues occur.
Prior to this invention much research had been done with Group B Streptococci in attempt to isolate and purify the antigenic serological determinants of this bacteria. These attempts to obtain pure antigenic material resulted in the finding that each of the serotypes of Group B Streptococcus mentioned above has associated with it antigenic material reactive with Group B serological determinants (Group B antigen) as well as antigenic material reactive with the particular serotype serological determinant (Type antigen). It is known that with various process manipulations the Type antigens can be separated from the cell wall of the bacteria and then separated from the Group antigens. Both of these separations, according to the prior art, resulted in Group antigens which were not immunologically pure and, e.g., Type III, antigens which had immunological purity but did not have all the characteristics required to produce a desirable vaccine. Both the Type III and Group B antigenic serological determinants were found to be polysaccharides. As used herein, "serological determinants" means materials which are present in blood and have antigenic sites.
The prior methods of obtaining the antigens were directed to extraction from the intact cell or cell walls with hydrochloric acid, trichloracetic acid (TCA), hot formamide, or EDTA extraction followed by disruption of the bacterial cells by glass beads, enzyme digestion, and alcohol precipitation. These processes ultimately resulted in a "pure" Type III antigen. The Group B antigen resulting from these processes was generally associated with serotype antigenic activity such as Type III. None of these processes of the prior art is satisfactory for isolating both pure Type III antigen and pure Group B antigen, having the properties required to use in a vaccine with desirable characteristics. The Type III antigen is important since it can be used in a vaccine to immunize against the most serious diseases caused by the Group B Streptococcus.
It is thus apparent that there is a need for a method to produce pure Group B Streptococcus antigens and pure Type III Streptococcus antigens in such a form that they will be suitable for vaccines for humans to protect against infections caused by all serotypes of Group B Streptococci and Group, Type III Streptococcus.